We thank the Agency for Science, Technology and Research (A*STAR), Singapore for funding this research.

The treatment of animals was performed in accordance with the IACUC and NACLAR guidelines and approved by the animal department ((http://www.brc.astar.edu.sg/index.php?sectionID-11). 
NOTE: Neurosphere cultures were prepared from the forebrain of embryonic (E14) C57BL/6 mice as previously described5.
1. Culture of Clonal Sample Neurospheres (Day 1)
<ol>
	<li>Prepare NSC growth medium by combining Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient F-12 (DMEM/F12) medium with B27 supplement (final concentration: 1x), EGF (final concentration: 20 ng/mL), bFGF (final concentration:10 ng/mL) and penicillin/streptomycin (final concentration:1x).</li>
	<li>Dissociate E14.5 mouse neurospheres in 1 mL&#160;NSC growth medium and 1 mL&#160;0.05 N Sodium Hydroxide for 7 min at RT. Pipette cells up and down occasionally to keep cells in suspension. Add 1 mL&#160;of 0.05 N Hydrochloric acid to neutralize the alkali. 
	NOTE: Neurosphere cells are seeded at 20,000 cells/mL in a 10 cm culture dish and are cultured for 5 d at 37 &#176;C, 5% CO2. The expected yield after&#160;is about 10 million cells per 10 cm dish. These cells are then dissociated and seeded at clonal density as described in steps 1.2 and 1.3, respectively.
	<ol>
		<li>Perform cell count using Trypan Blue staining and a hemocytometer.</li>
	</ol>
	</li>
	<li>Seed single E14.5 mouse neurosphere cells at a density of 50 cells/mL&#160;or below in 100 &#181;L&#160;NSC growth medium in a 96-well dish. Incubate the cells at 37 &#176;C, 5% CO2 for 1 w to generate clonal neurospheres. 
	NOTE: Clonality is ensured during neurosphere formation at this density as the cells do not contact each other.</li>
</ol>
2. Culture of Neurospheres for Conditioned Medium (Day 3)
<ol>
	<li>Dissociate E14.5 mouse neurospheres as described in step 1.2.</li>
	<li>Seed single cells from E14.5 mouse neurospheres at a density of 20,000 cells/mL&#160;in a 10 mL NSC growth medium in a 10&#160;cm culture dish. Incubate the cells at 37 &#176;C, 5% CO2 for 5 d to generate bulk cultured neurospheres.</li>
</ol>
3. Preparation of Neurosphere Conditioned Medium (Day 8)
<ol>
	<li>Prepare coating solution by mixing 700 &#181;L of 0.01% Poly-L-Lysine (PLL), 70 &#181;L&#160;of 1 mg/mL Laminin and 6,230 &#181;L of Phosphate Buffered Saline (PBS).</li>
	<li>Coat a 10 cm culture dish with 7 mL&#160;of PLL/Laminin solution for 2 h&#160;at 37 &#176;C.</li>
	<li>Using a 10 mL&#160;serological pipette, transfer all the bulk cultured neurospheres to a 15 mL&#160;conical centrifuge tube. Centrifuge the neurospheres at 171 x g for 1 min at RT and remove the supernatant completely.</li>
	<li>Prepare NSC differentiation medium by combining DMEM/F12 medium with B27 (final concentration:&#160;1x) and Fetal Bovine Serum (FBS) (final concentration: 0.5%)&#160;Add 10 mL&#160;of NSC differentiation medium to neurospheres. Pipette up and down a few times to resuspend the neurospheres.</li>
	<li>Aspirate PLL/Laminin from the 10 cm culture dish and wash once with 10 mL&#160;PBS. Transfer all the neurospheres to the PLL/Laminin-coated 10&#160;cm culture dish. Incubate O/N at 37 &#176;C, 5% CO2 for the neurospheres to adhere to the dish.</li>
</ol>
4. Determination of NFU of Sample Neurospheres (Day 8)
<ol>
	<li>Count the number of neurospheres (seeded on day 1; section 1) formed in each well under a microscope. 
	NOTE: Determine the number of neurospheres formed per 100 cells plated, which gives the NFU.</li>
</ol>
5. Transfer of Sample Neurospheres to 50-well Chambered Coverslip (Day 8)
<ol>
	<li>Coat each well of a 50-well chambered coverslip with 10 &#181;L&#160;of PLL/Laminin coating solution for 2&#160;h&#160;at 37 &#176;C. 
	NOTE: Prepare coating solution as described in step 3.1.</li>
	<li>Prepare 3 x 50 mL&#160;conical centrifuge tubes containing 30 mL&#160;of PBS each. Using sterile forceps, remove the coated 50-well chambered coverslip. Dip the chambered coverslip into the first tube of PBS, followed by the second and then the third tube of PBS, to wash off the coating solution.</li>
	<li>Aspirate any remaining liquid from the wells of the chambered coverslip.</li>
	<li>Gently pipette all medium and neurospheres from each well of the 96-well dish and transfer it&#160;to a single 10 cm culture dish.</li>
	<li>Under a microscope, pick a sample neurosphere using a P10 micropipette fitted with a 10 &#181;L&#160;pipette tip. Ensure that only a single neurosphere is picked each time by setting the pipette column to 2 &#181;L. Use a new pipette tip for each neurosphere.</li>
	<li>Transfer the neurosphere onto the center of a well of the coated 50-well chambered coverslip.</li>
	<li>Repeat steps 5.5 and 5.6 until each well of the chambered coverslip contains a neurosphere. 
	NOTE: The neurospheres may be picked under a microscope placed in a laminar flow hood or on a benchtop.</li>
	<li>Add 10 &#181;L&#160;of NSC differentiation medium to each well of the chambered coverslip. Place the chambered coverslip in a 10 cm culture dish and pipette PBS along the edges of the dish to prevent drying of the NSC differentiation medium. Place up to two chambered coverslips in one 10 cm culture dish. Incubate the chambered coverslip (contained in the 10&#160;cm culture dish) overnight at 37 &#176;C, 5% CO2.</li>
</ol>
6. Differentiation of Sample Neurospheres (Day 9)
<ol>
	<li>Transfer the differentiation medium from the bulk cultured neurospheres (from section 3) to a 15 mL&#160;conical centrifuge tube. Ensure that 2-3 mL&#160;of differentiation medium remains in the culture dish to prevent adhered cells from detaching. Using a 20 mL&#160;sterile syringe, pass the differentiation medium through a 0.2 &#181;m filter to remove any cells or debris.</li>
	<li>Transfer the filtered differentiation medium back to the 10&#160;cm culture dish containing the adhered cells.</li>
	<li>Using sterile forceps, gently place the chambered coverslip onto the differentiation medium in an inverted manner such that the sample neurospheres are facing downwards. Incubate the chambered coverslip with the differentiation medium for 3 d at 37 &#176;C, 5% CO2. 
	NOTE: The sample neurospheres are in contact with the differentiation medium but not in contact with the differentiating cells beneath.</li>
</ol>
7. Fixing of Differentiated Neurospheres (Day 12)
<ol>
	<li>Gently remove the chambered coverslip from the 10 cm culture dish using sterile forceps. Gently dip the coverslip into PBS to wash off the conditioned medium.</li>
	<li>Peel off the silicone gasket from the coverslip. Perform this gently and slowly to avoid breaking the coverslip. Take note of the side where the differentiated neurospheres are adhered.</li>
	<li>Pipette 1 mL&#160;of 4% paraformaldehyde (PFA) onto a piece of paraffin film cut to the size of the coverslip. Gently place the coverslip on the paraffin film with the neurospheres facing downwards in contact with the PFA. Fix the neurospheres for 20 min at RT. 
	Caution: PFA is toxic both in liquid and vapor form and is combustible. Avoid inhalation of vapor and contact with skin and eyes. Always handle PFA in a ventilated fume hood.</li>
	<li>Pipette 1 mL&#160;of PBS onto a piece of paraffin film. Wash the neurospheres by gently placing the coverslip with the neurospheres facing downwards in contact with PBS for 10 min at room temperature.</li>
	<li>Repeat step 7.4 two more times.</li>
</ol>
8. O4 Staining of Differentiated Neurospheres (Day 12)
<ol>
	<li>Prepare 3% bovine serum albumin (BSA) in PBS. Pipette 1 mL&#160;of the 3% BSA onto a piece of paraffin film. Block the neurospheres by gently placing the coverslip (with the neurospheres facing down) in contact with the BSA for 30 min at RT.</li>
	<li>Pipette 1 mL&#160;of mouse anti-O4 IgM antibody (1:300 dilution in the 3% BSA) onto a piece of paraffin film and place the coverslip on the paraffin film with the neurospheres facing downwards in contact with the antibody. Allow O4 staining to occur for 2 h&#160;at RT.</li>
	<li>Wash the coverslip twice with PBS as described in step 7.4.</li>
	<li>Pipette 1 mL&#160;of green fluorescent dye conjugated-anti-mouse IgM secondary antibody (1:500 dilution in the 3% BSA) onto a piece of paraffin film and place the coverslip on the paraffin film with the neurospheres facing downwards in contact with the antibody. Leave for 1 h&#160;at RT in the dark. 
	NOTE: Perform immunostaining in a dark environment from this step onwards.</li>
	<li>Wash the coverslip twice with PBS as described in step 7.4.</li>
</ol>
9. Tuj1 and Glial Fibrillary Acidic Protein (GFAP) Staining of Differentiated Neurospheres (Day 12)
<ol>
	<li>Prepare permeabilizing solution by adding Triton-X 100 (final concentration:&#160;0.5%) to the 3% BSA. Pipette 1 mL&#160;of permeabilizing solution onto a piece of paraffin film. Place coverslip on the paraffin film with the neurospheres facing downwards in contact with the permeabilizing solution for 20 min at RT.</li>
	<li>Wash the coverslip twice with PBS as described in step 7.4.</li>
	<li>Pipette 1 mL&#160;of mouse anti-Tuj1 IgG2a antibody (1:500 dilution in the&#160;3% BSA) and rabbit anti-GFAP IgG antibody (1:1,000 dilution in the 3% BSA) onto a piece of paraffin film and place coverslip on the paraffin film with the neurospheres facing downwards in contact with the antibodies. Allow Tuj1 and GFAP staining to occur for 2 h at RT. 
	NOTE: Both antibodies can be prepared as a single solution.</li>
	<li>Wash the coverslip twice with PBS as described in step 7.4.</li>
	<li>Pipette 1 mL&#160;of red fluorescent dye conjugated-anti-mouse IgG2a (1:500 dilution in the 3% BSA) and far red fluorescent dye conjugated-anti-rabbit IgG (1:500 dilution in the 3% BSA) secondary antibodies onto a piece of paraffin film and place coverslip on the paraffin film with the neurospheres facing downwards in contact with the antibodies. Leave for 1 h at RT. 
	NOTE: Both antibodies can be prepared as a single solution.</li>
	<li>Wash the coverslip twice with PBS as described in step 7.4.</li>
	<li>Dilute 4&#39;,6-diamidino-2-phenylindole (DAPI) stock solution to 300 nM in PBS. Pipette 1 mL&#160;of DAPI onto a piece of paraffin film and place coverslip on the paraffin film with the neurospheres facing downwards in contact with DAPI for 2 min at RT for nuclei staining.</li>
	<li>Wash the coverslip once with PBS and twice with deionized water as described in step 7.4. Mount the coverslip on a glass slide using a suitable mounting medium and allow to dry O/N at RT.</li>
</ol>
10. Imaging of Differentiated Neurospheres (Day 13)
<ol>
	<li>Image neurospheres under a confocal microscope using a 40X. Use the lasers at 488 nm, 594 nm and 647 nm to detect oligodendrocytes, neurons and astrocytes, respectively.</li>
	<li>Determine the percentage of unipotent, bipotent and tripotent neurospheres. 
	NOTE: Potency is determined by the ability of the neurosphere to differentiate into the three neural lineages - oligodendrocytes, neurons and astrocytes5,14,15,26. Oligodendrocytes are identified by positive staining for O4 (section 8). Neurons are identified by positive staining for Tuj1 (section 9). Astrocytes are identified by positive staining for GFAP (section 9). A unipotent neurosphere differentiates into only one of the lineage and should positively stain for either O4, GFAP or Tuj1. A bipotent neurosphere differentiates into any two lineages and should positively stain for O4 and GFAP or O4 and Tuj1 or GFAP and Tuj1. A tripotent neurosphere differentiates into all three lineages and should positively stain for O4, GFAP and Tuj1.</li>
	<li>Determine NSC frequency in the population of interest by using the following formula: 
	NSC frequency = (NFU x % of tripotent neurospheres) / 100%.</li>
</ol>

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The authors declare no competing financial interests.

We describe hee the enumeration of NSCs under clonal conditions. Critical steps include the use of fresh NSC growth and differentiation medium, and also the use of freshly prepared PLL/Laminin solution to coat the chambered coverslips, as well as the culture dishes. This ensures optimal growth and differentiation conditions of the neurospheres. The use of good quality antibodies to effectively detect the neurons and glia, as well as the selective picking of clonal neurospheres that are not in contact with other neurospheres, are critical. Furthermore, it is important to ensure the picked neurospheres do not dry&#160;up. It is recommended to add 10 &#181;L&#160;of differentiation medium to each well after picking every 10 neurospheres. It is important to use one chambered coverslip in one 10 cm dish per sample to avoid cross talk between neurospheres from different samples. If two coverslips (each containing neurospheres from different samples) are placed in the same 10 cm dish, neurospheres from one coverslip might release factors that may alter the propensity of neurospheres in the other coverslip to differentiate into specific lineages. Two coverslips in the same 10 cm dish may also result in mix&#160;up between samples.
In the event the NFU or the NSC frequency is lower than expected, ensure that low passage number neurospheres are used. It is also important that healthy low passage neurospheres are used for generating conditioned medium. Next, if the neurospheres are cultured in wells with small diameter, such as in 96-well dish, then it will be difficult to maneuver and pick neurospheres using a micropipette. In this case, transfer the neurospheres to a larger culture dish, such as a 6-well dish, prior to picking. Some of the picked neurospheres may lift off from the chambered coverslip during culture and immunostaining. Minimizing the movement of the chambered coverslip during culture, and less intensive washing during immunostaining, would help to avoid detachment of neurospheres.
Initial studies quantified NSCs using only the NFA7,11,13. However, the NFA overestimates the NSC frequency as both NSCs and early NPs generate neurospheres5. Louis et al. developed another method to quantify NSCs known as the NCFCA21,22. The NCFCA involves culturing single cells in a collagen based matrix to ensure clonality, and it was shown that neurospheres above 2 mm in diameter are formed by NSCs only. Similar to the NCFCA, clonality is ensured throughout this protocol. This is achieved by generating neurospheres at clonal density and differentiating the neurospheres in chambered coverslips. The use of chambered coverslips confers a number of advantages. The chambered coverslip allows each sample neurosphere to differentiate without having physical contact with other sample neurospheres, thereby ensuring clonality during differentiation. The chambered coverslip can be placed suspended on the differentiation medium to allow the sample neurospheres to be continuously conditioned and to ensure maximum differentiation. In the absence of conditioned medium and serum during differentiation, survival of the neurospheres decreases and the neurospheres mostly generate astrocytes (Tham M and Ahmed S, unpublished). In addition, the immunostained neurospheres can be conveniently stored for long period of time and the chambered coverslips can be mounted directly on the microscope glass slides. Additionally, imaging of the immunostained neurospheres is made easy as one can quickly locate the neurosphere in the small chambered well, capture an image, and move on to the next well.
We have determined the NSC frequency in E14.5 mouse neurosphere culture using both the protocol described in this manuscript27 and NCFCA28. The NSC frequency in E14.5 mouse neurospheres determined by both methods are comparable. The NCFCA enumerates NSCs that are multipotent and have long-term self-renewal capacity. Since the NSC frequency from our method is comparable to that from NCFCA, the readout from our protocol does not seem to overestimate the NSC frequency. The NCFCA requires three weeks to be completed and mainly involves cell plating and medium replenishment. The protocol described here requires 13 days to perform and involves a different series of steps, e.g., neurosphere picking and immunostaining. This protocol could serve as an alternative method to enumerate NSCs. Researchers can use both NCFCA and this protocol to verify NSC frequency in their samples.
One limitation of this protocol is that a series of important steps have to be all performed on day 8. The preparation of the conditioned medium, determination of NFU of sample neurospheres, and transfer of sample neurospheres to the 50-well chambered coverslip have to be performed on day 8. One might take time to complete these steps. In addition, it requires practice to perform these steps in an efficient manner.
Neurospheres have been widely used to study NSC function and behavior. However, neurospheres consist of both NSCs and NPs. Hence, it is important to enrich NSCs from neurospheres to study NSC biology. Currently, cell surface markers, dye exclusion property as well as cell size and granularity have been used to enrich for NSCs6,7,9-13,29,30. This protocol could be used to quantify the enrichment of NSCs by potential NSC markers, and to facilitate the search for a definitive NSC marker. Four recent studies have used this protocol to enumerate NSC frequency5,14,15,26.

Neural stem cells (NSCs) are cells of the central nervous system (CNS) that can self-renew and are multipotent. NSCs are first specified during development of the embryonic forebrain and continue to persist in the adult brain at specific regions such as the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus (DG) of the hippocampus. A number of NSC lines are being used in clinical trials for treatment of stroke and other neurological diseases such as Batten&#39;s disease1.
NSCs and neural progenitors (NPs) are propagated in culture as floating 3-dimensional spheroid structures called neurospheres. The neurosphere culture system was developed by Reynolds and Weiss in the early 1990s, when they found that embryonic and adult cortical cells could divide in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF)2-4. Neurospheres consist of a heterogeneous mix of cells comprising of subclasses at different developmental stages5. It is challenging to specifically study NSCs using data obtained from neurospheres due to the presence of NPs. Hence, it is crucial to enrich NSCs from neurospheres. At present, four main methods have been used to enrich for NSCs in vitro. First is the use of cell surface markers. Lewis-X (LeX) and CD133 (also known as Prominin1) are the most prominent cell surface NSC markers6-9. Syndecan-1, Notch-1 and Integrin-beta1 are other surface proteins that enrich for NSCs10. Second is the dye exclusion. It has been shown that the side-population cells, which have the unique ability to pump out the fluorescent DNA-binding dye Hoechst 33342, are enriched for NSCs11. Third is the use of morphological selection. It has been demonstrated that cells with increased cell size and granularity harbor more NSCs than their counterparts5,12,13. Fourth is the addition of NSC survival factors to the culture medium. It has been shown that addition of Chondroitin sulphate proteoglycan and Apolipoprotein E enhances NSC survival and thus increases NSC frequency14,15. Although many markers including transcription factors have been associated with NSCs16,17, none of these markers are able to enrich NSCs to purity. Due to the lack of definitive NSC markers, it remains a challenge to quantify NSCs and distinguish them from NPs in vitro.
Initial studies used the neurosphere formation assay (NFA) to quantify NSCs7,11,13. In this assay, dissociated cells are cultured to form neurospheres and the number of neurospheres generated for every 100 cells plated is determined. This value is termed as the Neurosphere Formation Unit (NFU). The NFU equals the NSC frequency if all neurospheres arise from NSCs. However, it was shown that the NFU overestimates the NSC frequency as neurospheres are formed by both NSCs and early NPs5. Thus, it is inaccurate to enumerate NSCs solely based on neurosphere formation. It could be possible to quantify NSCs based on their ability to self-renew, proliferate extensively and generate multipotent neurospheres.
NSCs, which are EGF and bFGF responsive, usually survive for at least ten passages and thus display extensive self-renewal capacity in culture4,18-20. NPs, which are&#160;EGF and bFGF responsive as well, could also generate neurospheres for a few passages but not for an extended period of time. Hence, it has been widely accepted that bona fide NSCs can be enumerated with fair accuracy based on neurosphere formation for at least ten passages. In most studies, however, self-renewal is usually measured based on secondary or tertiary neurosphere formation due to the long experimental time required for ten passages. Hence, the secondary NFA can be used to broadly compare the self-renewal ability between populations, but cannot be used to accurately enumerate NSC frequency.
NSCs have a greater proliferative ability compared to NPs. This property of NSCs was used by Louis et al. to develop an assay for NSC enumeration &#8212; the neural colony-forming cell assay (NCFCA)21,22. In this assay, single cells are cultured for 3 weeks in a collagen-containing semisolid matrix. Under these culture conditions, it was shown that cells that form neurospheres above 2 mm in diameter are tripotent and can self-renew for long term. These cells are defined as NSCs. Therefore, the NSCs are effectively distinguished from NPs.
NSCs have the ability to differentiate into astrocytes, oligodendrocytes and neurons. For differentiation of neurospheres, growth factors are removed and serum is added to the culture medium.&#160;If all three neural lineages are observed in a neurosphere, then the cell that initiated that neurosphere is an NSC. However, the differentiation assay has some limitations. First, the culture conditions used for differentiation may not be optimal for generation of all three neural lineages. In fact, in a single neurosphere differentiation process, significant cell death occurs and mostly astrocyte generation occurs (Tham M and Ahmed S, unpublished). Second, there is the issue of clonality. For accurate enumeration of NSCs, formation and differentiation of neurospheres have to be performed under clonal conditions, where each neurosphere arises from a single cell. In bulk suspension cultures, aggregation occurs at both cellular and neurosphere levels23-25. Hence, it is possible for each neurosphere to arise from multiple cells or neurospheres, which complicates neurosphere counting and evaluation of multipotency. Recent evidence shows that aggregation of cells does not occur at low plating density of 0.5 cells/&#181;L&#160;or below, and when culture plates are not moved during neurosphere formation15. Thus culturing cells at such low density would ensure clonality.
Currently, the NCFCA is the most commonly used method to distinguish NSCs from NPs and enumerate NSC frequency. The NCFCA, however, requires a relatively long&#160;time of three weeks. Here, we describe a protocol to enumerate NSC frequency based on the ability of NSCs to form multipotent neurospheres. This protocol takes only 13 days to perform. The NCFCA ensures clonality as the collagen matrix prevents movement of the neurospheres. The culture conditions used in this protocol also allows clonality to be maintained throughout. For instance, the use of 50-well chambered coverslips ensures that the neurospheres will differentiate without contacting each other. Furthermore, we use conditioned medium that supplies neurotrophic factors during differentiation to maximize the differentiation potential of the neurospheres (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 ml conical centrifuge tubes&#160;</td>
			<td>BD</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical centrifuge tubes&#160;</td>
			<td>BD</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>20 ml sterile syringe&#160;&#160;</td>
			<td>BD</td>
			<td>300141</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml sterile syringe&#160;</td>
			<td>BD</td>
			<td>300144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)&#160;</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Tuj1 IgG2a antibody&#160;</td>
			<td>Covance</td>
			<td>MMS-435P-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFAP IgG antibody</td>
			<td>Dako</td>
			<td>Z033401</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer&#160;</td>
			<td>Hausser Scientific</td>
			<td>3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope fitted to laminar flow hood&#160;</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Marienfeld-superior</td>
			<td>1000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-O4 IgM antibody&#160;</td>
			<td>Merck Millipore</td>
			<td>MAB345</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydromount</td>
			<td>National Diagnostics</td>
			<td>HS-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse</td>
			<td>TS100-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>NuAire</td>
			<td>NU-543</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture dishes</td>
			<td>NUNC</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm culture dishes&#160;</td>
			<td>NUNC</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin film</td>
			<td>Parafilm</td>
			<td>PM999</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (EGF)&#160;</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human basic fibroblast growth factor (bFGF)&#160;</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;m filter&#160;</td>
			<td>Sartorius Stedim</td>
			<td>16534-K</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.45 &#181;m filter&#160;</td>
			<td>Sartorius Stedim</td>
			<td>16555-K</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 N Sodium hydroxide (NaOH)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 N Hydrochloric acid (HCl)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (PLL)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA) powder&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) powder&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>50-well chambered coverslips&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C7735</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate with heat function&#160;</td>
			<td>Stuart</td>
			<td>UC152</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Fisher Scientific</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>1x phosphate buffered saline without Ca2+ and Mg2+ (DPBS)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgM antibody&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21042</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti-mouse IgG2a&#160; antibody&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21135</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 goat anti-rabbit IgG antibody&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31573</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6&#8217;-diamidino-2-phenylindole dihydrochloride (DAPI)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>D3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;RT1 75002383</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>Thermo Fisher Scientific</td>
			<td>10006021</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilated fume hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pH meter&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

enumeration of neural stem cells using clonal assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

We have used this protocol to assess the ability of LeX, a known cell surface NSC marker7, to enrich for NSCs. Data from this study will be used to illustrate how NSCs are enumerated using the clonal assays in the protocol. E14.5 mouse neurosphere cells were incubated with anti-LeX antibody. Subsequently, cells that express LeX (LeX+) and cells that do not express LeX (LeX-) were seeded at 50 cells/mL&#160;by Fluorescence-Activated Cell Sorting (FACS) and left to generate clonal neurospheres for 1 wk. Time-lapse imaging shows that the neurospheres generated at 50 cells/mL&#160;are clonal (Figure 2). The NFU for LeX+ and LeX- cells was determined. It is shown that LeX+ cells have a significantly higher neurosphere formation capability compared to LeX- cells (Figure 3g). The neurospheres generated were then differentiated under clonal conditions, subsequently immunostained and imaged (Figure 3a-e). LeX+ cells generate significantly more tripotent neurospheres than LeX- cells (Figure 3f). The NSC frequency was then calculated based on the NFU and % of tripotent neurospheres (Figure 3g). Selection based on LeX, enriches NSC frequency by more than 14&#160;fold, validating LeX as a NSC marker. We have also determined the NSC frequency of E14.5 mouse neurospheres using this protocol27 and NCFCA28. Both methods report comparable NSC frequency of about 2% in E14.5 mouse neurospheres.
<img alt="Figure 1" src="/files/ftp_upload/54456/54456fig1.jpg" /> 
Figure 1. Protocol for Neurosphere Differentiation under Clonal Conditions. Sample neurospheres grown under clonal conditions are individually placed in each PLL/Laminin-coated well of a 50-well chambered coverslip, and allowed to adhere O/N. On the same day, bulk cultured neurospheres are plated in differentiation medium in a PLL/Laminin-coated 10 cm dish, and allowed to adhere O/N. The next day, conditioned medium from the bulk cultured neurospheres is filtered and placed back in the 10 cm dish. The sample neurospheres are then inverted onto the conditioned medium and allowed to differentiate for 3-4 days28. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54456/54456fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54456/54456fig2.jpg" /> 
Figure 2. Growth of a Clonal Neurosphere. Cells derived from E14.5 neurospheres were dissociated and seeded at 50 cells/mL&#160;in 96-well dish. Individual cells were tracked for 6 d&#160;by time-lapse imaging. Images of one such cell growing into a neurosphere is shown. Note that the neurosphere maintains clonality. (a) 0 days; (b) 1.19.45; (c) 2.95.37; (d) 3.32.76; (e) 4.46.53; (f) 5.61.44; where the first number refers to the day, second number refers to the fraction of the day, and third number refers to the fraction of the h.&#160;10X&#160;magnification. Scale bar, 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54456/54456fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54456/54456fig3.jpg" /> 
Figure 3. Differentiation of Clonal Neurospheres and Enumeration of NSC Frequency. Single neurospheres were picked and differentiated in 50-well chambered coverslip for 3 d. A representative image of a tripotent neurosphere stained with (a) DAPI and immunostained for (b) GFAP, (c) Tuj1 and (d) O4. (e) Shows the overlay of (a) - (d). Scale bar, 65 &#181;m. (f) The % of unipotent, bipotent and tripotent neurospheres generated by LeX+ and LeX- cells (mean &#177;SEM; n = 3). (g) NSC frequency in LeX+ and LeX- cells calculated as the product of NFU and % of tripotent neurospheres. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54456/54456fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Enumeration of Neural Stem Cells Using Clonal Assays at JoVE.com

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

enumeration-of-neural-stem-cells-using-clonal-assays

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.2K Views.  Agency for Science, Technology and Research (A*STAR). Neural stem cells (NSCs) refer to cells which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Video: Enumeration of Neural Stem Cells Using Clonal Assays

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.